https://scholars.lib.ntu.edu.tw/handle/123456789/87861
標題: | 應用體細胞核移植技術在家畜育種及生物醫學之研究(1/3) | 作者: | 吳信志 | 關鍵字: | 體細胞;複製猪;核移植;體外成熟;猪卵母細胞;間葉幹細胞;Somatic Cells;Nuclear Transfer;Cloned pigs;In Vitro Maturation;Porcine Oocytes;Mesenchymal stem cells | 公開日期: | 2005 | 出版社: | 臺北市:國立臺灣大學動物科學技術學系暨研究所 | 摘要: | 本計畫第一年目標擬先以基因顯微注射法產製綠色螢光(enhanced green fluorescentprotein, EGFP)基因轉殖猪,作為第二年單株骨髓間質幹細胞分離之用。另持續進行優良種猪體細胞株之建立、種源保存、猪胚體外培養系統之建立和體細胞複製猪試驗。自卵巢表面濾泡 (直徑2~8mm) 收集卵丘卵母細胞複合體,培養於NCSU-23 成熟培養液中42-44 h,挑選排出第一極體之卵母細胞進行去核,再將單一之成纖維母細胞 (fibroblast)或間葉幹細胞細胞 (mesenchymal stem cell,MSC) 注射至已去核之卵母細胞內,重組胚經不同方式激活,包括 (1) 單次電擊激活(electroporation, EP, 10 sec at 5 V AC followedby a 1 x 30 μsec pulse at 2.2 kV/cm DC) 後,配合細胞骨架鬆弛劑 cytochalasin B, CB, 10μg/mL) 合併蛋白質合成抑制劑(cycloheximide, CHX, 10 μg/mL) 處理,或 (2)於單次電擊後,以蛋白質轉磷酸酶抑制劑(6-dimethylaminopurine, 6-DMAP, 2mM) 處理。結果顯示,以相同方式激活後,成纖維細胞作為供核細胞者囊胚率較高於以間葉幹細胞者 (37 vs. 34%, 28 vs. 24%),而以EP+ 6-DMAP 激活處理之分裂率及囊胚率(88-89%, 34-37%)均較高於以EP+CB+CHX 處理者(75-80%, 24-28%),其中以MSC 為供核細胞進行核轉置,並以EP+CB+CHX 激活之處理組,其囊胚率(24%)顯著低於以EP+6-DMAP 激活處理者(34-37%,p<0.05)。結果顯示,由MSC 所形成之重組胚體外發育能力較低於由成纖維細胞形成者,而電激後添加6-DMAP 進行化學激活處理可顯著改善重組胚之分裂及囊胚率。以上述試驗結果為基礎,嘗試以電激活配合6-DMAP 激活條件進行核移置胚之產製,期能提高複製猪之產製效率,試驗係以優良種公猪之耳皮膚成纖維母細胞當作供核細胞進行核移植之試驗,再次成功複製九頭優良種公猪,此結果證實產製效率已由0.5 % (4/795)提高至4 % (9/223),同窩可分娩九頭複製猪,已接近可產業化程度。 此外,以顯微注射EGFP 基因生產不同品系之綠色螢光小鼠,經基因注射後之原核胚,儘速移置回同期化處理之代理孕母體內,待懷孕期滿,以 PCR 檢測小鼠是否帶有外源基因並觀察其螢光表現。經分別移置326 及60 個FVB 及ICR鼠胚至12 和 2 隻代理孕母體內,並產下68及15 隻小鼠。以PCR 分析確定有9 (13 %) 及4 (27 %) 隻攜帶EGFP 基因,其中會表現綠色螢光者為6 (9 %, 2♂/ 4♀) 及4 (27 %, 2♂/2♀) 隻。相同轉殖基因亦被以猪胚原核基因顯微注入方式成功獲得二頭EGFP 基因轉殖猪胎兒,經長波長(365 nm)紫外光偵測後證實均可表現EGFP。表現EGFP 之小鼠均具有性腺遺傳能力,其子代有不同比例 (25-50 %)可表現EGFP。結果顯示,小鼠原核胚經注射EGFP 基因及胚移置後,可成功產下不同品系之基因轉殖小鼠,並可表現綠色螢光且具穩定之性腺遺傳能力。進一步分離綠色螢光轉基因小鼠純系之骨髓間葉幹細胞。以長波長(365 nm)紫外光偵測綠色螢光蛋白質基因在小鼠個體中之表現。結果顯示,各組織器官除脾臟、肺臟與骨組織外,其他器官如:心臟、肌肉、腦、肝、睪丸、腸道、腎、眼球及胰臟等均高度表達綠色螢光。源自股骨與脛骨的骨髓間葉幹細胞,經螢光顯微鏡檢查結果證明此等幹細胞確有表達綠色螢光之事實,進一步將猪及小鼠之骨髓間葉幹細胞於特定培養條件下進行誘導分化之試驗,已確認可分化成脂肪細胞。綜合上述,由MSC 所形成之重組胚體外發育能力較低於由成纖維細胞形成者,而電激後添加6-DMAP 處理可顯著改善重組胚之分裂率、囊胚率及產製複製猪之效率。本研究自綠色螢光小鼠骨髓成功分離獲得純系間葉幹細胞,可提供以猪為模式進行臨床前細胞治療、基因治療或組織工程等極具研究應用潛力之細胞來源。 The purposes of this study in the first year was to proceed with the cryopreservation of ear skin fibroblast cells derived from elite pigs’ herds are able to march forward continuously for cloning of elite pigs as well as to generate of EGFP (enhanced green fluorescent protein) transgenic pigs harboring EGFP marker gene by pronuclear microinjection for isolation of mescenchymal stem cells (MSC) of single clone derived from their bone marrow. In our results, primary fibroblast cells of top-performance individuals were established from skin samples taken from ear tissues. So far, derived somatic cells from over 50 elite pigs containing golden couples had been cryopreserved in liquid nitrogen tank. In addition, pig oocytes derived from ovaries of sow, cumulus-oocyte complexes were matured in vitro for 42–44 h, and oocytes with 1st polar body were selected for enucleation and reconstructed embryos could successfully activated by electrical activation and the electrical pulse combined with CB+ CHX or 6-DMAP would greatly improve the rates of cleavage to 80-90% and blastocyst formation rate to 30-40%. To study the effects of different donor cell types and activation methods on the subsequent development of porcine reconstructed embryos produced by somatic cell nuclear transfer (SCNT). A single suspended fibroblast or mesenchymal stem cell (MSC) was injected into enucleated ooplasm, and reconstructed embroys were activated by one of following methods: (1) electrical pulse (EP, 10 sec at 5 V AC followed by a 1 x 30 µsec pulse at 2.2 kV/cm DC) combined with 10 µg/mL cytochalasin B and 10 µg/mL cycloheximide for 4 h (EP+ CB+ CHX), or (2) EP combined with 2 mM 6-DMAP for 4 h (EP+ 6-DMAP). After activation treatments, the reconstructed embryos were thoroughly washed and cultured for 4 days in NCSU-23 medium. Results showed that the percentage of blastocyst formation in these treatments using the same activation method were greater in Fibroblast groups than in MSC groups (37 vs. 34%, 28 vs. 24%). The rates of cleavage and blastocyst formation were better in the groups treated with EP+ 6-DMAP (88-89%, 34-37%) than EP+ CB+ CHX (75-80%, 24-28%). Base on upper result, we generated successfully 9 cloned piglets in one litter by nuclear transfer of somatic cell derived from ear tissue of top-performance boar from core of artificial insemination. The efficiency of generating cloned piglet is elevated to 4 % (9/223) from 0.5 % (4/795). This result indicated the probability of this cloning technique for commercialization. Additionally, enhanced green fluorescent protein (EGFP) gene was introduced into mouse genome of FVB and ICR strain embryos by pronuclear microinjection. A total of 326 FVB and 60 ICR survival embryos were transferred into 12 and 2 recipient mice, respectively, and 68 FVB and 15 ICR pups were born. Nine (13 %) FVB and four (27 %) ICR mice were confirmed to be transgenic mice by PCR and Southern blotting analysis. Among them, six FVB (9 %, 2♂/ 4♀) and four ICR (27 %, 2♂/ 2♀) TG mice could express EGFP fluorescent and exhibit stable integration of the transgene in their germ line. As the experiments of EGFP transgenic mice, two transgenic fetuses of pig were produced and the EGFP expression pattern is similar to the transgenic mouse. The EGFP expression pattern of transgenic mice and pig fetuses was analysis by 365 nm UV light. Exclusion of spleen, lung and bone tissue, other organs (heart, muscle, brain, liver, testis, gut, kidney, eyes and pancreas etc.) highly expressed EGFP in transgenic mice and pig fetuses. Bone marrow was flushed out from femurs and tibias of transgenic candidates, the near-to confluence fibroblastic colony were obtained under the conditions of 2 X 106/cm2 of the starting culture and the incubation time of 72 hours. Expression of EGFP in isolated MSCs of transgenic pig fetuses and mice were detected by using fluorescent microscope. The EGFP MSCs derived from transgenic pig fetuses and mice were preliminary isolated from the transgenics in this study. Progressively, the MSCs were successfully conducted to give rise to adipocyte under defined culture conditions. In conclusions, our results indicates that the porcine reconstructed embryos using MSC as donor cell exhibit the poorer developmental potential in vitro, and the activation method of EP+ 6-DAMP was sufficient for cloned embryos to develop to term in our studies. The cell populations will be available in preclinical studies including cell therapy, gene therapy, tissue engineering fields and application in pig model. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/16473 | 其他識別: | 932317B002021 | Rights: | 國立臺灣大學動物科學技術學系暨研究所 |
顯示於: | 動物科學技術學系 |
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